CRISPR/Cas9-mediated gene knockout in human adipose stem/progenitor cells.
Mandl M., Ritthammer H., Ejaz A., Wagner SA., Hatzmann FM., Baumgarten S.
Laboratory Study, published in Adipocyte (2020) — summary generated from the PubMed abstract.
Evidence from laboratory and animal studies provides groundwork for understanding mechanisms and potential before human studies continue.
- Level A · Stronger Clinical Evidence
- Level B · Emerging clinical evidence with positive signals
- Level C · Early human research exploring benefits
- Level D · Scientific groundwork from lab and animal studies
- Emerging · Emerging topic under active research
This page is generated from the PubMed record. The Thai description is an automated summary of bibliographic fields and the abstract, not a full translation, and is not medical advice.
- Study type
- Laboratory Study
- Journal
- Adipocyte (2020)
- Country
- United States
- Reported sample size
- —
- Source database
- PubMed
- PMID
- 33070670
- PMCID
- PMC7575003
- DOI
- 10.1080/21623945.2020.1834230
- Citations
- 5
Abstract (original English)
The CRISPR/Cas9 system is a powerful tool to generate a specific loss-of-function phenotype by gene knockout (KO). However, this approach is challenging in primary human cells. In this technical report, we present a reliable protocol to achieve a functional KO in the genome of human adipose stem/progenitor cells (ASCs). Using Sprouty1 ( SPRY1 ) as a model target gene for a CRISPR/Cas9 mediated KO, we particularize the procedure including the selection of the CRISPR/Cas9 target sequences and the employment of appropriate lentiviral vectors to obtain a functional gene KO. The efficiency of CRISPR/Cas9 to mutate the SPRY1 gene is determined by a PCR-based mutation detection assay and sequence analysis. Effects on mRNA and protein levels are studied by RT-qPCR and Western blotting. In addition, we demonstrate that CRISPR/Cas9 mediated SPRY1 KO and gene silencing by shRNA are similarly effective to deplete the Sprouty1 protein and to inhibit adipogenic differentiation. In summary, we show a reliable approach to achieve a gene KO in human ASCs, which could also apply to other primary cell types. Abbreviations: ASC: Adipogenic Stem/Progenitor Cell; Cas: CRISPR-associated system; CRISPR: Clustered Regularly Interspaced Palindromic Repeat; gDNA: Genomic DNA; GOI: Gene of interest; gRNA: Guide RNA; NHEJ: Non-homologous end joining; Indel: In sertion/ Del etion; PAM: Protospacer adjacent
What this study does not prove
- • This study does not prove SVF is an approved treatment or a replacement for standard care.
- • This is preclinical work; animal or laboratory results cannot be applied to humans.
Evidence level
Evidence from laboratory and animal studies provides groundwork for understanding mechanisms and potential before human studies continue.
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